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Isolation of Exosomes from Semen for in vitro Uptake and HIV-1 Infection Assays
从精液中分离外来体用于体外摄取和HIV-1感染测定

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Abstract

Exosomes are membranous extracellular nanovesicles of endocytic origin. Exosomes are known to carry host and pathogen-derived genomic, proteomic, lipidomic cargos and other extraneous molecules. Exosomes are secreted by diverse cell types into the extracellular milieu and are subsequently internalized by recipient neighboring or distal cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. Exosomes facilitate intercellular communication, modulate cellular phenotype, and regulate microbial pathogenesis. We have previously shown that semen exosomes (SE) inhibit HIV-1 replication in various cell types. Here, we describe detailed protocols for characterizing SE. This protocol can be adapted or modified and used for evaluation of other extracellular vesicles of interest.

Keywords: Semen(精液), Exosomes(外来体), Extracellular(细胞外), Vesicles(囊泡), Prostasomes(前列腺体), HIV(HIV)

Background

Exosomes are membranous nanovesicles originating as a result of inward budding of endosomal membranes within the late endosomal compartment of a multitude of cell types (Simons and Raposo, 2009). Exosomes are released by many cell types (Iglesias et al., 2012) into the extracellular milieu and are found in biological fluids including blood (Kaur et al., 2014) urine (Li et al., 2013) saliva (Madison et al., 2015) and breast milk (Madison et al., 2014; Naslund et al., 2014). Human semen contains a heterogenous population of nanovesicles (Madison et al., 2014; Madison et al., 2015) produced by tissues of the male genital tract including prostate secretory acinar cells (Sahlen et al., 2002) and epididymal epithelial cells (Frenette et al., 2010) as well as cells of the vasa deferentia, testes, and the vesicular glands (Renneberg et al., 1997; Sullivan et al., 2005). The variability in the cells that secret exosomes is reflected in the composition and function of exosomes. Thus, exosomal cargo composition and function are regulated by many factors including the type and condition of the originating cell (Raposo and Stoorvogel, 2013), cellular environment, and for in vivo derived exosomes; the condition of the donor (Welch et al., 2017). Released exosomes when taken up by target cells transfer their cargo, including proteins (Iglesias et al., 2012; Charrier et al., 2014), miRNA (Shtam et al., 2013; Ong et al., 2014), and mRNA (Tomasoni et al., 2013; Madison et al., 2014; Madison et al., 2015) to the target cells. As a result, exosomes are known to be involved in modulation of host immune response (Kaur et al., 2014; Vojtech et al., 2014), and regulation of microbial pathogenesis (Li et al., 2013; Arenaccio et al., 2014; Madison et al., 2014; Naslund et al., 2014; Vojtech et al., 2014; Madison et al., 2015).
   While progress has been made in the field of exosome biology, many protocols are contradictory in the most effective and efficient method of characterizing exosomes (Taylor and Shah, 2015). Here, we provide a detailed protocol for evaluating the function and physical properties of semen exosomes (Madison et al., 2014; Madison et al., 2015). This protocol lays the groundwork for evaluating other functional activities of semen exosomes, and for evaluating exosomes from other sources.

Materials and Reagents

  1. Pipette tips (any brand)
  2. 15 polypropylene conical plastic tubes (DOT SCIENTIFIC, PerformR®, catalog number: 416-PG )
  3. 50 ml polypropylene conical plastic tubes (DOT SCIENTIFIC, PerformR®, catalog number: 451-PG )
  4. 12 well tissue culture plate (CELLTREAT Scientific Products, catalog number: 229112 )
  5. 5 ml polystyrene round-bottom tubes (Corning, Falcon®, catalog number: 352052 )
  6. Microscope coverslip, 18 mm (Fisher Scientific, FisherbrandTM, catalog number: 12-545-100 )
  7. Coverslips (VWR, catalog number: 48382-041 )
  8. Microscope slides (Fisher Scientific, catalog number: 22-034-486)
  9. 1.7 m microcentrifuge tube (DOT SCIENTIFIC, catalog number: RN1700-GMT )
  10. 96 well solid white flat-bottom polystyrene microplates (Corning, catalog number: 3917 )
  11. 96 well tissue culture plate (CELLTREAT Scientific Products, catalog number: 229196 )
  12. Disposable cuvettes (Eppendorf, catalog number: Z605050 )
  13. 1 ml disposable syringes (BD, catalog number: 309659 )
  14. 96 well tissue culture dishes
  15. U937 human monocytic cell line
  16. TZM-bl human vaginal epithelial cell line
  17. Jurkat human T lymphocyte cell line
  18. V428 (HPV-16 E6/E7 transformed human vaginal epithelial cell line)
  19. VK2 (HPV-16 E6/E7 transformed human vaginal epithelial cell line)
  20. Human semen
  21. Cell-free HIV-1 virus stock, replication competent
  22. ExoQuick (System Biosciences)
  23. Phosphate buffered saline, DPBS 1x; without CaCl2 and MgCl2 (Thermo Fisher Scientific, GibcoTM)
  24. Liposomes (Lipofectamine 2000) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  25. PKH67Green fluorescent kit (Sigma-Aldrich, catalog number: MINI67 )
  26. PKH26Red fluorescent kit (Sigma-Aldrich, catalog number: MINI26 )
  27. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM)
  28. Quick Start Bradford Protein Assay Kit 1 (Bio-Rad Laboratories, catalog number: 500-0201 )
  29. Roswell Park Memorial Institute (RPMI) 1640 (Thermo Fisher Scientific, GibcoTM)
  30. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM)
  31. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM)
  32. L-glutamine (Thermo Fisher Scientific, GibcoTM)
  33. Keratinocyte serum free media (KSFM) (Thermo Fisher Scientific)
  34. Human recombinant Epidermal Growth Factor 1-53 (Thermo Fisher Scientific)
  35. Bovine pituitary extract (BPE) (Thermo Fisher Scientific)
  36. 0.25% trypsin-EDTA, phenol red dissociation reagent (Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056 )
  37. Collagen, type 1 from rat tail (Sigma-Aldrich, catalog number: C3867 )
  38. Paraformaldehyde (2%) (Fisher Scientific, catalog number: T353-500 )
  39. Vectashield antifade reagent with DAPI (Vector Laboratories, catalog number: H-1200 )
  40. Exosome-human CD63 Isolation/Detection (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10606D )
  41. Albumin, bovine fraction V (BSA) (RPI, catalog number: A30075-100.0 )
  42. Anti-human CD63-FITC (BioLegend, catalog number: 353005 )
  43. Aldehyde/Sulfate latex beads (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: A37301 )
  44. MHC-II monoclonal antibody
  45. Isotype control antibody (mouse IgG1)
  46. Glycine (RPI, catalog number: G36050-500.0 )
  47. Anti-human CD63-PE (BioLegend, catalog number: 353003 )
  48. Triton-X-100 (Sigma-Aldrich, catalog number: X100 )
  49. Acetylthiocholine chloride (Sigma-Aldrich, catalog number: A5626 )
  50. 5,5’-Dithiobis 2-nitrobenzoic acid (Sigma-Aldrich, catalog number: D8130 )
  51. Sodium carbonate, anhydrous (RPI, catalog number: S25025-500.0 )
  52. NuPAGE LDS sample buffer (4x) (Thermo Fisher Scientific, NovexTM, catalog number: NP0008 )
  53. NuPAGETM NovexTM 4-12% Bis-Tris Protein Gels, 1.5 mm, 10-well (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0335PK2 )
  54. NuPAGE MOPS SDS running buffer (20x) (Thermo Fisher Scientific, NovexTM, catalog number: NP0001 )
  55. Pierce Silver Stain Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 24612 )
  56. RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
  57. RNase-Free DNase Set (QIAGEN, catalog number: 79254 )
  58. High-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4368814 )
  59. QuantiFast SYBR Green PCR Kit (QIAGEN, catalog number: 204054 )
  60. Agarose (RPI, catalog number: A20090 )
  61. Ethidium bromide solution (Bio-Rad Laboratories, catalog number: 1610433 )
  62. 1x TAE buffer
  63. Steady-Glo (Promega, catalog number: E2510 )
  64. NuPAGE sample reducing agent (10x) (Thermo Fisher Scientific, NovexTM, catalog number: NP0004 )
  65. MES (Sigma-Aldrich, catalog number: M2933 )
  66. 1 N NaOH (Avantor Performance Materials®, J.T.Baker®, catalog number: 563502 )
  67. Distilled water (any brand)
  68. Exosome-depleted FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
  69. MTT reagent (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: M6494 )
  70. Nonidet P-40 substitute (RPI, catalog number: N59000 )
  71. Hydrochloric acid (HCl), ACS reagent, 37% (Sigma-Aldrich, catalog number: 258148 )
  72. Isopropanol (Fisher Scientific, catalog number: A416-4 )
  73. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM)
  74. Exosome-depleted FBS (see Recipes)
  75. Lysing sample buffer for protein footprint (see Recipes)
  76. Storage buffer (see Recipes)
  77. MES buffer (see Recipes)
  78. FACS wash buffer (see Recipes)
  79. MTT reagent (see Recipes)
  80. MTT solvent (see Recipes)

Equipment

  1. Pipettes (any brand)
  2. Sterile tweezers (any brand)
  3. Centrifuge (Eppendorf, model: 5415 D)
  4. Ultracentrifuge (Beckman Coulter, model: Optima L-90K )
  5. SW40Ti rotor (Beckman Coulter, catalog number: 331362 )
  6. Polyallomer centrifuge tubes 14 x 89 mm for SW41Ti rotor (Beckman Coulter, catalog number: 344059 )
  7. 37 °C, 5% CO2 cell culture incubator (NuAire, model: NU-5510 )
  8. FACSCalibur flow cytometer (BD) (see Notes for laser and filter specifications)
  9. FACSVerse flow cytometer (BD) (see Notes for laser and filter specifications)
  10. FACSAria flow cytometer (BD) (see Notes for laser and filter specifications)
  11. Laser scanning confocal microscope (Nikon Instruments, model: Eclipse TE2000 )
  12. Magnetic separator for 1.7 ml tubes
  13. Rotating mixer
  14. Microplate reader (Tecan Trading, model: Infinite® M200 Pro )
  15. DynaPro Nanostar (Wyatt Technologies, model: DynaPro Nanostar )
  16. NanoSight LM10 (Malvern Instruments, model: NanoSight LM10 )
  17. NanoDrop spectrophotometer (Thermo Fisher Scientific)
  18. SW60 Ti rotor (Beckman Coulter, catalog number: 335649 )
  19. Polyallomer centrifuge tubes 11 x 60 mm for SW60Ti rotor (Beckman Coulter, catalog number: 355636 )
  20. 7500 fast real-time PCR system (Thermo Fisher Scientific, model: 7500 Fast Real-time PCR System )
  21. Luminometer (BioTek Instruments, model: Synergy H1 Hybrid Reader )
  22. Laminar flow hood
  23. SW32 Ti rotor (Beckman Coulter, model: 369650 )
  24. Polyallomer centrifuge tubes 25 x 89 mm for SW32Ti rotor (Beckman Coulter, catalog number: 344058 )
  25. pH meter (any brand)
  26. Gel running tank(Thermo Fisher Scientific, NovexTM, model: XCell SureLock® Mini-Cell , catalog number: EI0001)
  27. Sephacryl S300-HR 16/60 gel filtration prepacked column (GE Healthcare catalog number: 17-1167-01 )
  28. BioLogic DuoFlow Workstation (Bio-Rad Laboratories)
  29. BioLogic BioFrac fraction collector (Bio-Rad Laboratories)
  30. Gel electrophoresis horizontal apparatus (Bio-Rad Laboratories, model: Wide Mini-Sub Cell GT Cell, catalog number: 1704468EDU )
  31. Dry Block Heater for microcentrifuge tubes (Thermo Fisher Scientific)

Software

  1. FlowJo analysis software (TreeStar)
  2. BioLogic DuoFlow software version 5.3 (Bio-Rad Laboratories)
  3. Dynamics software (Wyatt Technology)
  4. NTA software (Malvern Instruments)

Procedure

  1. Acquisition of human semen samples
    This study utilized existing human specimens (semen) and therefore is not human subjects’ research. The samples were discarded from routine examinations and not linked to any identifiers.
    1. Collect semen by dry manual stimulation and ejaculation into sterile 15 ml polypropylene conical tubes.
    2. Store samples at room temperature for 30 min to promote liquefaction and then centrifuge for 10 min at 1,000 x g at 4 °C to pellet spermatozoa.
    3. Remove seminal plasma from spermatozoa-containing pellets. Pellets can be discarded or stored for downstream analysis.
    4. Store seminal plasma samples at -80 °C until required for exosome purification by ExoQuick or ultracentrifugation. We have found no discernable difference between the two methods of purification.

  2. ExoQuick purification of exosomes
    1. Thaw seminal plasma samples before centrifuging at 2,000 x g for 15 min at 4 °C in a 50 ml conical tube. Transfer supernatant to a new tube and centrifuge again at 10,000 x g for 30 min at 4 °C in a 50 ml conical tube to pellet cellular debris.
    2. Place clarified seminal plasma in a fresh 50 ml conical tube and add ExoQuick at a ratio of 4:1 (clarified seminal plasma/ExoQuick). Mix by inversion, and incubate at 4 °C overnight (12-24 h).
    3. Centrifuge the clarified seminal plasma/ExoQuick mixture at 1,500 x g for 30 min at 4 °C.
    4. Remove the supernatant (exosome free clarified seminal plasma and ExoQuick) and repeat centrifugation at 1,500 x g for 10 min at 4 °C without resuspension of the pellet.
    5. Remove the residual supernatant.
    6. Resuspend the exosome pellet in PBS to 1/10 of the original volume of seminal plasma, quantify protein concentration by Bradford assay, and aliquot.

  3. Ultracentrifugation purification of exosome
    1. Dilute clarified seminal plasma 50% in PBS, and ultracentrifuge at 100,000 x g for 2 h at 4 °C to pellet exosomes using a SW41Ti rotor.
    2. Wash exosome pellets in PBS three times with ultracentrifugation at 100,000 x g for 30 min per wash (1.5 h). A volume of PBS should be used that completely fills the ultracentrifuge tubes to avoid collapse of the tubes.
    3. Resuspend exosomes in PBS to 1/10 of the original volume of seminal plasma, quantify protein concentration by Bradford assay, and aliquot.

  4. Fluorescent labeling of exosomes, liposome controls or PBS controls
    1. Fluorescently label PBS control, Lipofectamine 2000 derived liposome control, or exosomes using PKH67Green or PKH26Red kits according to manufacturer’s instructions with the following modifications: Add 1 mg of purified exosomes or liposome control or an equivalent volume of PBS to 250 µl of PBS and mix with 250 µl of Diluent C in SW41Ti ultracentrifuge tubes.
    2. Add 4 µl of PKH67Green or PKH26Red lipophilic dye to 500 µl of Diluent C in a separate tube at room temperature (RT) in the dark and perform all subsequent steps at RT in the dark.
    3. Combine the tube containing dye and Diluent C with the tube containing exosomes or controls in PBS and Diluent C in the SW41Ti ultracentrifuge tube. Rapidly mix by manual pipetting. Incubate for 5 min in the dark at RT and vortex twice during the 5-min incubation.
    4. After the 5 min of incubation, add exosome-free FBS in a 1:1 ratio to the ultracentrifuge tube containing dye, exosomes or controls, and Diluent C. Incubate for 1 min to occupy unbound dye.
    5. Ultracentrifuge the solution at 100,000 x g for 30 min. Discard the supernatant.
    6. Resuspend the pellet containing dye-labelled exosomes or controls in PBS, transfer to another SW41Ti ultracentrifuge tube and wash three times by ultracentrifugation at 100,000 x g for 30 min per wash. After each wash, transfer the PBS resuspended pellet to a new SW41Ti ultracentrifuge tube to minimize transfer of unbound dye. A volume of PBS should be used that completely fills the ultracentrifuge tube to avoid collapse of the tube.
    7. After the final wash, remove the supernatant, and resuspend the pellet in PBS to the original volume of the exosomes. Quantify dye-labelled exosomes or controls using Bradford Protein Assay Kit, aliquot, and store in the dark at -80 °C until use.

  5. Cell culture preparation for internalization of fluorescent exosomes
    1. Grow human monocytic and lymphocytic cell lines in tissue culture dishes in RPMI supplemented with 10% exosome depleted FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 0.3 mg/ml L-glutamine in a 5% CO2 incubator at 37 °C.
    2. Grow TZM-bl vaginal epithelial cell lines in tissue culture dishes in DMEM supplemented with 10% exosome depleted FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 0.3 mg/ml L-glutamine in a 5% CO2 incubator at 37 °C.
    3. Grow VK2 and V428 vaginal epithelial cell lines in tissue culture dishes in keratinocyte serum free media supplemented with 0.1 ng/ml prequalified human recombinant Epidermal Growth Factor 1-53, 0.05 mg/ml BPE, 100 U/ml penicillin and 100 μg/ml streptomycin.
    4. Grow all cell types in 12 well tissue culture dishes. Seed cells at 1 x 105 cells per well in 1 ml volume of the correct cell culture media for each cell type (see Note 1).
    5. Expose cells to 25-100 μg/ml of red or green fluorescent exosomes or controls for 0, 3, 6, 9, 12 or 24 h incubation periods in a 5% CO2 incubator at 37 °C. The incubation of SE with cells should be completed in cell culture media. Final volume of SE incubation with cells will depend on the concentration of exosomes after fluorescent labeling.
    6. Wash TZM-bl, VK2 or V428 adherent cells three times in 1 ml PBS in the plate and detach the cells from the plate using 0.5 ml of 0.25% trypsin-EDTA dissociation reagent. Neutralize the trypsin with 0.5 ml 10% exosome free FBS in DMEM. Collect the cells from the wells and transfer to 5 ml polystyrene round-bottom tubes. Wash the cells three times in 1 ml PBS with 5 min centrifugations at 500 x g at 4 °C.
    7. Collect monocytic and lymphocytic suspension cells in 5 ml polystyrene round-bottom tubes. Treat with trypsin as described in step E5, and neutralize with 10% exosome free FBS in RPMI. Wash the cells three times in 1 ml PBS with 5 min centrifugations at 500 x g at 4 °C.
    8. Assess cellular uptake and exosome internalization kinetics utilizing FACS analysis or confocal microscopy.
      1. For FACS analysis:
        1. Fix cells for 15 min on ice with 300 μl 2% paraformaldehyde.
        2. Wash the cells two times in PBS with 5 min centrifugations at 500 x g in between each wash. Resuspend the cell pellets in 300 μl PBS.
        3. Analyze fluorescence using a FACSCalibur or FACSVerse flow cytometer (BD) to detect the PKH67Green (FL-1 or FITC channel, respectively) or PKH26Red (FL-2 or PE channel, respectively) transferred from exosomes to cells during fusion and uptake.
        4. Determine cellular frequency and fluorescence intensity using FlowJo analysis software (TreeStar) (Figure 1).


          Figure 1. Internalization of fluorescent exosomes by FACS. A. VK2 cellular uptake of PKH67 green labeled PBS vehicle or 100 μg/ml semen exosomes (SE) at 3- and 24-h post exposure. B. V428 cellular uptake of PK67 green labeled PBS vehicle or 25 or 100 μg/ml SE at 24-h post exposure. C. Jurkat, U937, and TZM-bl cellular uptake of PKH26 red or PKH67 green labeled PBS vehicle or 100 μg/ml SE at 24-h post exposure. The y-axis shows the forward scattering value (FSC) of the cell populations.

      2. For confocal microscopy analysis of exosome internalization:
        1. Coat coverslips with collagen by adding sterile, round 18 mm coverslips to the wells of a 12 well plate. Add 0.5 ml of 50 μg/ml collagen solution to the wells. Incubate the plate at 37 °C for 1 h, after which you move the plate to 4 °C for overnight incubation. After overnight incubation, remove the collagen solution and gently wash the coverslips in the wells three times with PBS. After the final wash, aspirate all the PBS and immediately proceed to step E8b.ii.
        2. Grow TZM-bl, VK2 or V428 adherent cells in 12 well tissue culture plates on top of collagen coated microscope cover slips, expose to fluorescent exosomes as described in step E4 and wash three times in PBS in the plate.
        3. Fix cells on the coverslips in the plate with 2% paraformaldehyde for 15 min with the plate on ice.
        4. Remove coverslips from the plate and add a drop of Vectashield antifade reagent to each coverslip.
        5. Mount coverslips down on microscope slides.
        6. Assess fusion, uptake and internalization kinetics of fluorescent red or green exosomes using laser scanning confocal microscopy. Representative images are shown in Madison et al ., 2015.

  6. Detection of surface exosomal markers
    Here we will describe detection of human CD63 in SE, but this protocol may be modified for detection of other common exosomal surface markers in SE. Human CD63 in SE is detected per the manufacturer’s instructions using the exosome-human CD63 isolation/detection kit from Invitrogen where after acquiring SE:
    1. Resuspend 25 μg of SE to a total volume of 100 μl in 0.1% BSA.
    2. Resuspend anti-CD63 coated magnetic beads by vortexing for 30 sec. Transfer 20 μl of beads to a microcentrifuge tube and wash the beads by adding 200 μl of 0.1% BSA and vortexing.
    3. Place the tube containing the beads on a magnet separator for 1 min. Discard the supernatant before removing the tube from the magnet.
    4. Add the SE solution to the washed beads and mix by pipetting.
    5. Incubate the SE/beads solution at 4 °C overnight (18-20 h) in a rotating mixer.
    6. Centrifuge the SE/beads solution for 3-5 sec.
    7. Wash the SE bound beads in 300 μl of 0.1% BSA and mix by pipetting before placing the tube on the magnet for 1 min and discarding the supernatant.
    8. Remove the tube from the magnet. Wash the SE bound beads in 400 μl of 0.1% BSA and mix by pipetting.
    9. Place the tube on the magnet for 1 min and discard the supernatant before removal from the magnet.
    10. Resuspend the SE bound beads in 300 μl of 0.1% BSA.
    11. Transfer 100 μl of the SE bound beads to a new microcentrifuge tube. Add 5 μl of anti-human CD63-FITC (BioLegend) and mix by pipetting.
    12. Incubate the SE-bounds beads and antibody for 60 min at room temperature in the dark on an oscillating plate.
    13. Wash the antibody stained SE-bound beads in 300 μl of 0.1% BSA and mix by pipetting. Place the tube on the magnet for 1 min, and discard the supernatant before removal of the tube from the magnet.
    14. Repeat the washing step twice before resuspending the SE-bound beads in 300 μl of 0.1% BSA. Transfer the resuspended SE-bound beads to a 5 ml polystyrene round-bottom tube before analysis on FACSVerse instrument and FlowJo (Tree Star) software (Figure 2).


      Figure 2. CD63 expression on SE. 25 μg SE or PBS vehicle were incubated overnight with α-CD63 coated magnetic beads to facilitate binding. Unbound SE was removed before staining of bead-bound SE with α-CD63-FITC and FACS analysis. O/N = overnight. The y-axis shows the forward scattering value (FSC) of the beads.

      Alternatively, common exosomal markers in SE may also be detected with the use of non-magnetic beads. Here we provide instructions for the use of latex beads to detect CD63 in SE.
      1. Per manufacturer instructions, 2.5 ml of resuspended, surfactant-free, 4-μm diameter, aldehyde/sulphate, latex beads (Invitrogen, Molecular Probes, hereafter referred to as latex beads) were washed twice in 10 ml of 0.025 M, pH 6.0 2-(N-morpholino) ethenesulfonic acid (MES) buffer with centrifugation at 3,000 x g for 20 min at 4 °C.
      2. Following the second and final wash, the latex beads were resuspended in 5 ml MES buffer.
      3. Incubate 100 µl latex beads with 100 µl of anti MHC-II MAb or isotype control antibody prepared in MES at a concentration of 1 mg/ml at room temperature overnight with gentle agitation.
      4. Sediment latex beads with conjugated antibody by centrifugation at 3,000 x g for 20 min at 4 °C.
      5. Remove supernatant (unbound antibody).
      6. Wash latex beads with conjugated antibody thrice in 1 ml PBS (0.1 M, pH 7.2) at 3,000 x g for 20 min at 4 °C.
      7. Resuspend latex beads in 100 µl of storage buffer (see Recipes).
      8. ExoQuick purified exosomes (100 μg) were incubated with *2 x 105 anti MHC-II or isotype control coated latex beads in a final volume of 100 μl PBS (0.1 M, pH 7.2) first for 15 min at room temperature followed by overnight at 4 °C with gentle agitation.
        *Note: Concentration of beads/ml differs by lot number.
      9. The reaction was stopped by 30 min incubation with PBS (0.1 M, pH 7.2) including 0.2% glycine to saturate any vacant sites on the latex beads.
      10. The exosome and antibody bound latex bead preparation was then washed thrice in FACS wash buffer (see Recipes).
      11. Exosomes coated beads were then incubated with the appropriate concentration of anti-CD63 conjugated to PE (BioLegend) or isotype control antibody for 1 h at room temperature in the absence of light followed by three washes in FACS buffer.
      12. The resulting immunofluorescence was analyzed by use of a FACSAria flow cytometer (BD) and FlowJo analysis software (TreeStar) (Figure 2).

  7. SE acetylcholinesterase activity
    1. Lyse 50 μg of SE in 2% Triton-X-100 at a 1:1 volumetric ratio.
    2. Add 5 μl of SE/Triton-X reaction to a 96-well flat bottom clear plate in replicates of three.
    3. Combine 1.25 mM acetylthiocholine chloride (Sigma-Aldrich) and 0.1 mM 5,5’-dithiobis 2-nitrobenzoic acid (Sigma-Aldrich) in a 1:1 ratio to reach a final volume of 100 μl per well. Add 100 μl of the mixed solutions to each of the exosome containing wells. Be sure the microplate reader has reached 37 °C before adding this solution to the wells as the reaction will start immediately after addition.
    4. Read absorbance at 450 nm on a microplate reader for a total of 30 min in 5 min intervals at 37 °C (Figure 3).


      Figure 3. Acetylcholine esterase activity of SE. 50 μg SE or PBS vehicle were lysed in Triton-X-100. AChE activity was measured at 5 min intervals for a total of 30 min. Error bars represent standard deviation.

  8. Dynamic Light Scattering (DLS) of SE
    1. Dilute 0.1 mg/ml of SE in 200 μl of PBS and analyze size by DynaPro NanoStar DLS (Wyatt Technologies) using a total volume of 150 μl in plastic cuvettes.
    2. Complete data analysis using Dynamics software.
    3. Use an average of ten measurements per exosome sample to determine radius, diameter, and %intensity (Figure 4).


      Figure 4. SE size and concentration estimation. A. Dynamic light scattering indicates approximate diameter of SE. 0.1 mg/ml of SE was used to measure the diameter of SE. Shown is the range of exosome diameters in the population. B. Representative image from NanoSight NTA video clip showing SE particles. C. Approximation of SE particles per ml of semen calculated from NanoSight estimation of concentration. D. Approximation of SE size by NanoSight. N = refers to the number of donors that were combined for exosome purification.

  9. SE NanoSight nanoparticle tracking analysis (NTA)
    1. Prepare control suspension containing uniformly sized (100 or 200 nm) polystyrene particles.
    2. Use the suspension to align the foci of the laser and microscope.
    3. Make serial dilutions of each SE specimen in PBS to a final volume of 0.5 ml.
    4. Inject 0.5 ml of diluted SE into NanoSight LM10 NTA using 1 ml disposable syringe.
    5. Analyzed individual members of the prepared serial dilutions until the raw concentration detected is within the recommended range for the instrument.
    6. Using the identified dilution, record three 30 sec videos for each specimen.
    7. Repeat the analysis 3 times using the same settling to ensure repeatable and accurate measurement.
    8. Complete post-acquisition analysis with NTA software to determine size and concentration of SE.
    9. View the results tab for i) total number of particles traced, ii) average number of particles, and iii) particle concentration.
    10. Calculate total concentration of exosomes per ml of semen accounting for the dilution factor used for NanoSight analysis (Figure 4). Obtain error bars by analyzing standard deviation of the 3 measurements of each sample.

  10. Fractionation of SE into membrane and luminal contents
    1. Treat 100 μg SE with 5-10x volume of 0.1 M sodium carbonate pH = 11.5. Mix by vortexing.
    2. Incubate for 30 min to 1 h at 4 °C. At this point, total protein from the lysed exosomes can be measured by NanoDrop spectrophotometer at 280 nm. Complete the following steps to separate lysed SE into membrane and luminal fractions.
    3. Transfer SE/sodium carbonate mix to SW60 Ti ultracentrifuge tubes. Bring up the volume with PBS to fill the tube.
    4. Ultracentrifuge at 150,000 x g for 1.5-2 h at 4 °C. The pellet contains the membrane fraction while the supernatant contains the luminal fraction.
    5. Remove the luminal ‘supernatant’ fraction. This can be concentrated by MW filter cutoffs before protein quantification by Bradford assay and downstream analysis or storage at -80 °C until use.
    6. Resuspend the membrane fraction in PBS to the original volume of SE before lysis. Quantify protein by Bradford assay, aliquot, and store at -80 °C or use in downstream analysis (Figure 5).


      Figure 5. Separation of SE into different fractions. SE can be fractionated into membrane and luminal components.

  11. SE protein footprint
    1. Lyse 5 μg of SE in 20 μl of PBS at a ratio of 3 μl NuPAGE lysing sample buffer (see Recipes) per 7 μl SE.
    2. Heat SE in sample buffer at 90 °C for 10 min.
    3. Load a maximum volume of 40 μl on a 10 well 1.5 mm NuPAGE 4-12% Bis-Tris gel.
    4. Run the gel at 200 V for ~50 min in NuPAGE 20x MOPS SDS running buffer diluted to 1x.
    5. Silver stain the gel following the manufacturer’s protocol (Pierce silver stain kit Thermo Scientific).

  12. Examination of RNA integrity and content of SE
    1. Use a starting concentration of at least 12 μg of SE for RNA extraction.
    2. Extract SE RNA using the Qiagen RNeasy kit per the manufacturer’s instructions and complete the optional DNase treatment using Qiagen RNase-free DNase set.
    3. Determine RNA concentration by NanoDrop spectrophotometer. To evaluate RNA integrity of SE, RNA can be analyzed by an Agilent BioAnalysis run using RNA 6000 Pico chips if the RNA concentration is < 50 ng/μl. If the RNA concentration is > 50 but < 500 ng/μl, the RNA can be evaluated using RNA 6000 Nano chips (Figure 6).


      Figure 6. RNA integrity of SE. RNA was extracted from SE and analyzed by Agilent Bioanalyzer.

    4. Use equivalent concentrations of RNA for cDNA synthesis using High-Capacity cDNA Reverse Transcription Kit (ABI).
    5. Use human gene specific primers to amplify CD9, CD63, and GAPDH or other genes of interest by Quantifast Sybr green technology (QIAGEN) and 7500 fast real-time machine.
    6. Visualize PCR amplicons on a 2% agarose gel by ethidium bromide staining. Representative images are shown in Madison et al ., 2014.

  13. SE inhibition of HIV-1 infectivity
    1. Grow TZM-bl cells in 96 well tissue culture dishes in DMEM supplemented with 10% exosome depleted FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 0.3 mg/ml L-glutamine in a 5% CO2 incubator at 37 °C.
    2. Preincubate 100 μg/ml SE or PBS vehicle with 8 RT units of HIV-1 virus for 1 h at 37 °C in DMEM supplemented with 10% exosome depleted FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 0.3 mg/ml L-glutamine. Preincubate supplemented DMEM with PBS vehicle as uninfected control. Mix before incubation by vortexing.
    3. Remove DMEM media from TZM-bl cells. Add 100 μl per well in triplicate of SE/HIV-1, PBS vehicle/HIV-1, or PBS vehicle/DMEM to TZM-bl cells.
    4. Incubate in a 5% CO2 incubator at 37 °C for 24 h.
    5. Access cell viability by MTT assay and HIV-1 infectivity by Steady-Glo luciferase assay (see Note 5; Figure 7)
      1. For MTT assay:
        1. Add 20 μl 5 mg/ml of MTT reagent to each well in replicates of three. Incubate for 3.5 h in a 5% CO2 incubator at 37 °C.
        2. Remove the media and MTT solution from the wells. Add 150 μl off MTT solvent (see Recipes) to each well and incubate for 15 min in the dark on an oscillating plate.
        3. Read absorbance at 590 nm on a microplate reader.
      2. For Steady-Glo luciferase assay:
        1. Remove DMEM media from TZM-bl cells. Add 100 μl of Steady-Glo luciferase reagent to each well in replicates of three, including uninfected control. Allow 5 min for cell lysis.
        2. Transfer 90 μl of lysed cells/Steady-Glo mixture to a solid white 96 well plate, avoiding the formation of bubbles.
        3. Read luciferase activity in a microplate luminometer.


          Figure 7. SE inhibition of HIV-1. Exosomes (100 μg/ml) or vehicle PBS were preincubated with 8 RT units HIV-1 NL4.3 virus for 1 h at 37 °C before infection of TZM-bl cells for 24 h. A. Infectivity measured by luciferase units; B. Viability is determined by MTT. TZM-bl cells were pretreated with exosomes (100 μg/ml) or vehicle PBS for 24 h before infection with 8 RT units HIV-1 NL4.3 virus for an additional 24 h. C. Infectivity read by luciferase units; D. Viability determined by MTT. Vehicle set as reference at 100% for infectivity and viability. Error bars are standard deviations.

Data analysis

  1. Each assay should be experimentally repeated at least three times with replicates of three per experiment to verify reproducibility.
  2. Average replicates of independent experiments to evaluate statistical significance. Where appropriate, compare results to vehicle control.
  3. Plot data using graphing software, such as Graphpad Prism. For analyses that are not represented graphically such as Bioanalyzer, protein footprint, and FACS analysis representative images may be shown.

Notes

  1. Cell culture
    When growing cells, to minimize evaporation, only plate cells in the inner wells of the plate and fill the outermost wells with PBS. We recommend plating cells the day before they will be used to allow the cells to normalize. Because SE alters cell viability at differing concentrations and in a cell-type dependent manner, it is important to evaluate cell cytotoxicity in all experiments involving cell treatments with SE (Madison et al., 2014; Madison et al., 2015). This is also important when evaluating HIV-1 infectivity as high concentrations of virus or SE may be cytotoxic to cells and influence analysis of results.
  2. Storage of exosomes
    We found that repeated freeze-thawing of exosomes decreases functional activity. We recommend that after isolation, to aliquot exosomes into individual microcentrifuge tubes (< 100 μg) before freezing for storage to retain functional activity.
  3. Discarding of supernatant
    When discarding supernatant after pelleting or washing exosomes, we recommend removal by pipette aspiration rather than inversion to ensure complete removal. However, exosome pellets may not always be easily observed, depending on the concentration used, and caution should be used to not dislodge the pellet.
  4. Sterility
    As often as feasibly possible, all experimental steps should be completed under a laminar flow hood to ensure an aseptic environment. Contamination may influence downstream analyses.
  5. HIV-1 infectivity
    Because TZM-bl cells contain background fluorescence, we recommend plating 10,000 cells per well in a 96 well format to minimize background luciferase expression. Depending on the cell line used, the HIV-1 isolate used may vary depending on the receptor/co-receptors expressed on that cell line. If evaluating infection in cells that are not a reporter cell line, infection may be evaluated by qRT-PCR using HIV-1 gene specific primers. Data may be confirmed by measuring intracellular or extracellular HIV-1 RT activity (Madison et al., 2014; Madison et al., 2015). All HIV-1 experiments must be carried out in accordance with biosafety training and laboratory environment requirements.
  6. SE-mediated inhibition of HIV-1
    SE also inhibits HIV-1 infection during a pretreatment model of infection. In this model, after growing the cells, SE is added to cells 24 h before virus infection, and remains inhibitory to infection. The HIV-1 inhibitory characteristic of SE is upheld in both the preincubation and pretreatment models with other cells lines such as: Jurkat, SUPT1, U937, PM1, THP-1, CEM, and PBLs (Madison et al., 2014; Madison et al., 2015).
  7. Flow cytometer specifications
    Laser and filter specifications of flow cytometer systems used in this protocol are included below.
    FACSCalibur flow cytometer (BD)
    Lasers: Air-cooled, argon-ion, 488 nm, 15 mW
    Emission detection:
                 FL1 530/30
                 FL2 585/42
                 FL3 670 LP
                 FL4 661/16
                 SSC 488/10
                 FSC 488/10
    FACSVerse flow cytometer (BD)
    Lasers:  Blue laser, 488 nm, 20 mW, beam spot size 9 x 63 μm
                 Red laser, 640 nm, 40 mW, beam spot size 9 x 63 μm
                 Violet laser, 405 nm, 40 mW, beam spot size 9 x 63 μm
    Emission detection:
                 FITC 527/32
                 PE 586/42
                 PerCp 700/54
                 APC 660/10
                 SSC 488/15
                 FSC 488/10
    FACSAria flow cytometer (BD)
    Lasers:  Coherent Sapphire, solid state, 488 nm, 20mW
                JDS Uniphase HeNe, air-cooled, 633 nm, 18mW
                Point Source Violet, solid state, 405 nm, 15 mW
    Emission detection:
                 FITC 530/30
                 PE 576/26
                 PerCp 695/40
                 APC 660/20
                 SSC 488/10
                 FSC 488/10

Recipes

  1. Exosome-depleted FBS
    Ultracentrifuge FBS at 100,000 x g for 2 h at 4 °C in SW32Ti ultracentrifuge tubes using SW32Ti rotor
    Collect supernatant and store at 4 °C for up to 1 week or at -80 °C for longer periods of time
  2. Lysing sample buffer for protein footprint
    Mix 250 μl NuPAGE 4x LDS sample buffer with 100 μl NuPAGE 10x reducing agent
    Add mixed buffer to SE samples at 3 μl buffer per 7 μl SE
  3. Storage buffer
    PBS (0.1 M, pH 7.2) and 0.1% glycine
  4. MES buffer
    Dissolve MES in distilled water for a concentration of 0.025 M. Determine pH with a pH meter, and adjust the pH with 1 N NaOH to 6.0
  5. FACS wash buffer
    1% exosome-depleted FBS in PBS
  6. MTT reagent
    Resuspend MTT reagent in PBS for final concentration of 5 mg/ml, per manufacturer’s instructions
  7. MTT solvent
    0.1% NP-40 and 4 mM HCl in isopropanol

Acknowledgments

This work was supported by the National Institute on Drug Abuse (NIDA) grant 1R01DA042348-01 (to CMO), National Institutes of Health (NIH) 5T32AI007533-18 (to JLW), and NIH T32 postdoctoral training grant in Infectious Diseases (to MNM), shared Instrumentation Grants 1S10RR025439-01 to the University of Iowa Central Microscopy Core facility, and Holden Comprehensive Cancer Center support grant P30CA086862. The authors are thankful to Aloysius Klingelhutz of the University of Iowa for providing V428 cells, to Bartholomey Konan and Melanie Freeman of the Reproductive Specialty Laboratory of Middle Tennessee and to Amy E.T. Sparks of the University of Iowa Hospitals and Clinics (UIHC) In Vitro Fertilization and Reproductive Testing Laboratory for providing pre-existing, de-identified human donor semen samples. We acknowledge the support of University of Iowa core facilities, including Central Microscopy, X-ray crystallography, and DNA core. We thank Sankar Baruah and Lokesh Gakhar of University of Iowa Crystallography Core Facility for help with Dynamic light scattering. The authors declare that they have no competing interests. MNM, JLW, and CMO wrote the paper. MNM and JLW contributed equally to this manuscript and are thus co-first authors. All authors reviewed the manuscript and approved the final version. Protocols described herein are adapted from our previously published works (Madison et al ., 2014 and 2015; Welch et al ., 2017).

References

  1. Arenaccio, C., Chiozzini, C., Columba-Cabezas, S., Manfredi, F., Affabris, E., Baur, A. and Federico, M. (2014). Exosomes from human immunodeficiency virus type 1 (HIV-1)-infected cells license quiescent CD4+ T lymphocytes to replicate HIV-1 through a Nef- and ADAM17-dependent mechanism. J Virol 88(19): 11529-11539.
  2. Charrier, A., Chen, R., Chen, L., Kemper, S., Hattori, T., Takigawa, M. and Brigstock, D. R. (2014). Exosomes mediate intercellular transfer of pro-fibrogenic connective tissue growth factor (CCN2) between hepatic stellate cells, the principal fibrotic cells in the liver. Surgery 156(3): 548-555.
  3. Frenette, G., Girouard, J., D'Amours, O., Allard, N., Tessier, L. and Sullivan, R. (2010). Characterization of two distinct populations of epididymosomes collected in the intraluminal compartment of the bovine cauda epididymis. Biol Reprod 83(3): 473-480.
  4. Iglesias, D. M., El-Kares, R., Taranta, A., Bellomo, F., Emma, F., Besouw, M., Levtchenko, E., Toelen, J., van den Heuvel, L., Chu, L., Zhao, J., Young, Y. K., Eliopoulos, N. and Goodyer, P. (2012). Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro. PLoS One 7: e42840.
  5. Kaur, S., Singh, S. P., Elkahloun, A. G., Wu, W., Abu-Asab, M. S. and Roberts, D. D. (2014). CD47-dependent immunomodulatory and angiogenic activities of extracellular vesicles produced by T cells. Matrix biology 37: 49-59.
  6. Li, J., Liu, K., Liu, Y., Xu, Y., Zhang, F., Yang, H., Liu, J., Pan, T., Chen, J., Wu, M., Zhou, X. and Yuan, Z. (2013). Exosomes mediate the cell-to-cell transmission of IFN-alpha-induced antiviral activity. Nat Immunol 14: 793-803.
  7. Madison, M. N., Jones, P. H. and Okeoma, C. M. (2015). Exosomes in human semen restrict HIV-1 transmission by vaginal cells and block intravaginal replication of LP-BM5 murine AIDS virus complex. Virology 482: 189-201.
  8. Madison, M. N., Roller, R. J., Okeoma, C. M. (2014). Human semen contains exosomes with potent anti-HIV-1 activity. Retrovirology 11: 102.
  9. Naslund, T. I., Paquin-Proulx, D., Paredes, P. T., Vallhov, H., Sandberg, J. K. and Gabrielsson, S. (2014). Exosomes from breast milk inhibit HIV-1 infection of dendritic cells and subsequent viral transfer to CD4+ T cells. AIDS 28(2): 171-180.
  10. Ong, S. G., Lee, W. H., Huang, M., Dey, D., Kodo, K., Sanchez-Freire, V., Gold, J. D. and Wu, J. C. (2014). Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer. Circulation 130(11 Suppl 1): S60-69.
  11. Raposo, G. and Stoorvogel, W. (2013). Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200(4): 373-383.
  12. Renneberg, H., Konrad, L., Dammshauser, I., Seitz, J. and Aumuller, G. (1997). Immunohistochemistry of prostasomes from human semen. Prostate 30(2): 98-106.
  13. Sahlen, G. E., Egevad, L., Ahlander, A., Norlen, B. J., Ronquist, G. and Nilsson, B. O. (2002). Ultrastructure of the secretion of prostasomes from benign and malignant epithelial cells in the prostate. Prostate 53(3): 192-199.
  14. Shtam, T. A., Kovalev, R. A., Varfolomeeva, E. Y., Makarov, E. M., Kil, Y. V. and Filatov, M. V. (2013). Exosomes are natural carriers of exogenous siRNA to human cells in vitro. Cell Commun Signal 11: 88.
  15. Simons, M. and Raposo, G. (2009). Exosomes--vesicular carriers for intercellular communication. Curr opin cell biolo 21(4): 575-581.
  16. Sullivan, R., Saez, F., Girouard, J. and Frenette, G. (2005). Role of exosomes in sperm maturation during the transit along the male reproductive tract. Blood Cells Mol Dis 35(1): 1-10.
  17. Taylor, D. D. and Shah, S. (2015). Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes. Methods 87: 3-10.
  18. Tomasoni, S., Longaretti, L., Rota, C., Morigi, M., Conti, S., Gotti, E., Capelli, C., Introna, M., Remuzzi, G. and Benigni, A. (2013). Transfer of growth factor receptor mRNA via exosomes unravels the regenerative effect of mesenchymal stem cells. Stem Cells Dev 22(5): 772-780.
  19. Vojtech, L., Woo, S., Hughes, S., Levy, C., Ballweber, L., Sauteraud, R. P., Strobl, J., Westerberg, K., Gottardo, R., Tewari, M. and Hladik, F. (2014). Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions. Nucleic Acids Res 42(11): 7290-7304.
  20. Welch, J. L., Madison, M. N., Margolick, J. B., Galvin, S., Gupta, P., Martínez-Maza, O., Dash, C. and Okeoma, C. M. (2017). Effect of prolonged freezing of semen on exosome recovery and biologic activity. Sci Rep 7: 45034.

简介

外来体是内膜起源的膜性胞外纳米囊。 已知外来载体携带宿主和病原体衍生的基因组,蛋白质组,脂质体载体和其他外来分子。 外来体由不同细胞类型分泌到细胞外环境中,随后被受体相邻细胞或远端细胞内化。 在内化后,外来体通过捐赠其载体和或激活各种信号转导途径来调节受体细胞,从而调节生理和病理生理过程。 外来体促进细胞间通讯,调节细胞表型和调节微生物发病机制。 我们以前表明精液外来体(SE)抑制各种细胞类型的HIV-1复制。 在这里,我们描述特征SE的详细协议。 该方案可以适应或修改,并用于评估感兴趣的其他细胞外小泡。

外来体是由许多细胞类型的晚期内体室内的内体膜向内发生的结果而引起的膜状纳米囊(Simons and Raposo,2009)。外来体被许多细胞类型(Iglesias等人,2012)释放到细胞外环境中,并且被发现在包括血液在内的生物流体中(Kaur等人,2014)尿(Liem等人,2013)唾液(Madison等人,2015)和母乳(Madison等人,2014; Naslund ,2014)。人类精液含有由包括前列腺分泌腺泡细胞在内的男性生殖道组织产生的纳米囊泡的异质群体(Madison等人,2014; Madison等人,2015) (Sahlen等人,2002)和附睾上皮细胞(Frenette等人,2010)以及vasa感染,睾丸和囊泡腺细胞( Renneberg等人,1997; Sullivan等人,2005)。秘密外来体细胞的变异性反映在外来体的组成和功能上。因此,外来货物组成和功能受许多因素的调节,包括原始细胞的类型和状况(Raposo和Stoorvogel,2013),细胞环境和体内衍生的外来体;供体的状况(Welch等人,2017)。由靶细胞吸收的释放的外来体转移其载体,包括蛋白质(Iglesias等人,2012; Charrier等人,2014),miRNA(Shtam et al。,2013; Ong等人,2014)和mRNA(Tomsoni等人,2013; Madison等人, ,2014; Madison等人,2015)到目标细胞。因此,已知exosomes参与宿主免疫应答的调节(Kaur等人,2014; Vojtech等人,2014),以及微生物调节发病机制(Li et al。,2013; Arenaccio等人,2014; Madison等人,2014; Naslund等人2014; Vojtech等人,2014年;麦迪逊等人,2015)。
虽然在外来体生物学领域取得了进展,但许多方案在表征外来体的最有效和最有效的方法上是矛盾的(Taylor and Shah,2015)。在这里,我们提供了一个详细的方案来评估精液外来体的功能和物理性质(Madison等人,2014;麦迪逊等人,2015)。该方案为评估精液外来体的其他功能活动以及从其他来源评估外来体奠定了基础。

关键字:精液, 外来体, 细胞外, 囊泡, 前列腺体, HIV

材料和试剂

  1. 移液器提示(任何品牌)
  2. 15个聚丙烯圆锥形塑料管(DOT SCIENTIFIC,PerformR ®,目录号:416-PG)
  3. 50ml聚丙烯圆形塑料管(DOT SCIENTIFIC,PerformR ®,目录号:451-PG)
  4. 12孔组织培养板(CELLTREAT Scientific Products,目录号:229112)
  5. 5 ml聚苯乙烯圆底管(Corning,Falcon ®,目录号:352052)
  6. 显微镜盖玻片18毫米(Fisher Scientific,Fisherbrand TM ,目录号:12-545-100)
  7. 盖片(VWR,目录号:48382-041)
  8. 显微镜幻灯片(Fisher Scientific,目录号:22-034-486)
  9. 1.7米微量离心管(DOT SCIENTIFIC,目录号:RN1700-GMT)
  10. 96孔固体白色平底聚苯乙烯微孔板(Corning,目录号:3917)
  11. 96孔组织培养板(CELLTREAT Scientific Products,目录号:229196)
  12. 一次性比色皿(Eppendorf,目录号:Z605050)
  13. 1 ml一次性注射器(BD,目录号:309659)
  14. 96孔组织培养皿
  15. U937人单核细胞系
  16. TZM-bl人阴道上皮细胞系
  17. Jurkat人T淋巴细胞细胞系
  18. V428(HPV-16 E6/E7转化人阴道上皮细胞系)
  19. VK2(HPV-16 E6/E7转化人阴道上皮细胞系)
  20. 人类精液
  21. 无细胞HIV-1病毒库,复制能力
  22. ExoQuick(系统生物科学)
  23. 磷酸盐缓冲盐水,DPBS 1x;不含CaCl 2和MgCl 2(Thermo Fisher Scientific,Gibco TM
  24. 脂质体(Lipofectamine 2000)(Thermo Fisher Scientific,Invitrogen TM,目录号:11668019)
  25. PKH67Green荧光试剂盒(Sigma-Aldrich,目录号:MINI67)
  26. PKH26Red荧光试剂盒(Sigma-Aldrich,目录号:MINI26)
  27. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM
  28. 快速入门Bradford蛋白测定试剂盒1(Bio-Rad Laboratories,目录号:500-0201)
  29. 罗斯韦尔公园纪念研究所(RPMI)1640(Thermo Fisher Scientific,Gibco TM
  30. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM
  31. 丙酮酸钠(Thermo Fisher Scientific,Gibco TM
  32. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM
  33. 角质形成细胞无血清培养基(KSFM)(Thermo Fisher Scientific)
  34. 人重组表皮生长因子1-53(Thermo Fisher Scientific)
  35. 牛垂体提取物(BPE)(Thermo Fisher Scientific)
  36. 0.25%胰蛋白酶-EDTA,酚红解离试剂(Thermo Fisher Scientific,Gibco TM,目录号:25200-056)
  37. 胶原,大鼠尾巴1型(Sigma-Aldrich,目录号:C3867)
  38. 多聚甲醛(2%)(Fisher Scientific,目录号:T353-500)
  39. Vectashield具有DAPI的抗褪色剂(Vector Laboratories,目录号:H-1200)
  40. Exosome-human CD63 Isolation/Detection(Thermo Fisher Scientific,Invitrogen TM,目录号:10606D)
  41. 白蛋白,牛馏分V(BSA)(RPI,目录号:A30075-100.0)
  42. 抗人CD63-FITC(BioLegend,目录号:353005)
  43. 醛/硫酸酯乳胶珠(Thermo Fisher Scientific,Molecular Probes TM,目录号:A37301)
  44. MHC-II单克隆抗体
  45. 同种型对照抗体(小鼠IgG1)
  46. 甘氨酸(RPI,目录号:G36050-500.0)
  47. 抗人CD63-PE(BioLegend,目录号:353003)
  48. Triton-X-100(Sigma-Aldrich,目录号:X100)
  49. 乙酰硫基胆碱氯化物(Sigma-Aldrich,目录号:A5626)
  50. 5,5'-二硫代双-2-硝基苯甲酸(Sigma-Aldrich,目录号:D8130)
  51. 无水碳酸钠(RPI,目录号:S25025-500.0)
  52. NuPAGE LDS样品缓冲液(4x)(Thermo Fisher Scientific,Novex TM,目录号:NP0008)
  53. NuPAGETM Novex TM 4-12%Bis-Tris蛋白凝胶,1.5mm,10孔(Thermo Fisher Scientific,Invitrogen TM,目录号:NP0335PK2)
  54. NuPAGE MOPS SDS运行缓冲液(20x)(Thermo Fisher Scientific,Novex TM,目录号:NP0001)
  55. Pierce Silver Stain Kit(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:24612)
  56. RNeasy Mini Kit(QIAGEN,目录号:74104)
  57. RNase-Free DNase Set(QIAGEN,目录号:79254)
  58. 大容量cDNA逆转录试剂盒(Thermo Fisher Scientific,Applied Biosystems TM,目录号:4368814)
  59. QuantiFast SYBR Green PCR Kit(QIAGEN,目录号:204054)
  60. 琼脂糖(RPI,目录号:A20090)
  61. 溴化乙锭溶液(Bio-Rad Laboratories,目录号:1610433)
  62. 1x TAE缓冲区
  63. Steady-Glo(Promega,目录号:E2510)
  64. NuPAGE样品还原剂(10x)(Thermo Fisher Scientific,Novex TM,目录号:NP0004)
  65. MES(Sigma-Aldrich,目录号:M2933)
  66. 1 N NaOH(Avantor Performance Materials ,J.T.Baker ,目录号:563502)
  67. 蒸馏水(任何品牌)
  68. Exosome-depleted FBS(Thermo Fisher Scientific,Gibco TM,目录号:26140079)
  69. MTT试剂(Thermo Fisher Scientific,Molecular Probes TM,目录号:M6494)
  70. Nonidet P-40替代品(RPI,目录号:N59000)
  71. 盐酸(HCl),ACS试剂,37%(Sigma-Aldrich,目录号:258148)
  72. 异丙醇(Fisher Scientific,目录号:A416-4)
  73. Dulbecco改良Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM
  74. 外来体消耗的FBS(参见食谱)
  75. 用于蛋白质足迹的裂解样品缓冲液(参见食谱)
  76. 存储缓冲区(请参阅配方)
  77. MES缓冲区(见配方)
  78. FACS洗涤缓冲液(参见食谱)
  79. MTT试剂(参见食谱)
  80. MTT溶剂(见配方)

设备

  1. 移液器(任何品牌)
  2. 无菌镊子(任何品牌)
  3. 离心机(Eppendorf,型号:5415 D)
  4. 超速离心机(Beckman Coulter,型号:Optima L-90K)
  5. SW40Ti转子(Beckman Coulter,目录号:331362)
  6. 用于SW41Ti转子的多聚物离心管14 x 89 mm(Beckman Coulter,目录号:344059)
  7. 37℃,5%CO 2细胞培养箱(NuAire,型号:NU-5510)
  8. FACSCalibur流式细胞仪(BD)(见激光和滤光片规格说明)
  9. FACSVerse流式细胞仪(BD)(见激光和滤光片规格说明)
  10. FACSAria流式细胞仪(BD)(见激光和滤光片规格说明)
  11. 激光扫描共焦显微镜(Nikon Instruments,型号:Eclipse TE2000)
  12. 1.7 ml管的磁选机
  13. 旋转搅拌机
  14. 酶标仪(Tecan Trading,型号:Infinite ® M200 Pro)
  15. DynaPro Nanostar(Wyatt Technologies,型号:DynaPro Nanostar)
  16. NanoSight LM10(Malvern Instruments,型号:NanoSight LM10)
  17. NanoDrop分光光度计(Thermo Fisher Scientific)
  18. SW60 Ti转子(Beckman Coulter,目录号:335649)
  19. 用于SW60Ti转子的聚对苯二甲酸酯离心管11 x 60 mm(Beckman Coulter,目录号:355636)
  20. 7500快速实时PCR系统(Thermo Fisher Scientific,型号:7500快速实时PCR系统)
  21. 发光计(BioTek Instruments,型号:Synergy H1 Hybrid Reader)
  22. 层流罩
  23. SW32 Ti转子(Beckman Coulter,型号:369650)
  24. 用于SW32Ti转子的聚对苯二甲酸酯离心管25 x 89 mm(Beckman Coulter,目录号:344058)
  25. pH计(任何品牌)
  26. 凝胶运行罐(Thermo Fisher Scientific,Novex TM,型号:XCell SureLock ® Mini-Cell,目录号:EI0001)
  27. Sephacryl S300-HR 16/60凝胶过滤预填柱(GE Healthcare目录号:17-1167-01)
  28. BioLogic DuoFlow工作站(Bio-Rad Laboratories)
  29. BioLogic BioFrac馏分收集器(Bio-Rad Laboratories)
  30. 凝胶电泳水平仪(Bio-Rad Laboratories,型号:Wide Mini-Sub Cell GT Cell,目录号:1704468EDU)
  31. 用于微量离心管的干式加热器(Thermo Fisher Scientific)

软件

  1. FlowJo分析软件(TreeStar)
  2. BioLogic DuoFlow软件版本5.3(Bio-Rad Laboratories)
  3. 动力软件(Wyatt Technology)
  4. NTA软件(Malvern Instruments)

程序

  1. 获取人类精液样品
    本研究利用现有的人体标本(精液),因此不是人类受试者的研究。样品从常规检查中丢弃,不与任何标识符相关。
    1. 通过干手动刺激和射精将精液收集到无菌的15ml聚丙烯锥形管中。
    2. 将样品在室温下储存30分钟以促进液化,然后在4℃下以1,000×g离心10分钟以沉淀精子。
    3. 从含有精子的颗粒中取出精浆。可以丢弃或储存颗粒用于下游分析。
    4. 将精液中的血浆样品储存在-80°C,直到ExoQuick进行外源体纯化或超速离心。我们发现两种纯化方法之间没有明显差异。

  2. ExoQuick净化外来体
    1. 解冻精液血浆样品,然后在400℃离心2000分钟,在4℃下在50ml锥形管中离心15分钟。将上清液转移到新管中,并在4℃下在10,000ml锥形管中以10,000xg再次离心30分钟以沉淀细胞碎片。
    2. 将澄清的精液血浆置于新鲜的50ml锥形管中,并以4:1的比例(澄清的精浆/ExoQuick)添加ExoQuick。通过倒置混合,并在4℃下孵育过夜(12-24小时)。
    3. 在4℃下将澄清的精浆等离子体/ExoQuick混合物以1,500×g离心30分钟。
    4. 取出上清液(不含外来物质的澄清精液等离子体和ExoQuick),并在4℃下以1,500 x g重复离心10分钟,而不会重新沉淀。
    5. 去除残留的上清液。
    6. 将PBS中的外来体颗粒重悬于原始体积的精浆中的1/10,通过Bradford测定法定量蛋白质浓度,并分批。

  3. 外植体超速离心纯化
    1. 在PBS中稀释澄清的精浆50%,并在4℃下以100,000×g超速离心2小时,以使用SW41Ti转子来沉淀外来体。
    2. 将PBS中的外来细胞颗粒洗涤三次,以100,000×g超速离心每次洗涤30分钟(1.5小时)。应使用一定体积的PBS,完全填充超速离心管,以避免管子塌陷。
    3. 将PBS中的外来体重悬于原始体积的精浆中的1/10,通过Bradford测定法定量蛋白质浓度,并分批。

  4. 荧光标记的外来体,脂质体对照或PBS对照
    1. 荧光标记PBS对照,Lipofectamine 2000衍生的脂质体对照或使用PKH67Green或PKH26Red试剂盒的外来体,根据制造商的说明书进行以下修改:将1mg纯化的外来体或脂质体对照或等体积的PBS加入到250μlPBS中,并与在SW41Ti超速离心管中加入250μl稀释剂C
    2. 在黑暗中室温(RT)下,在独立的管中,将5μlPKH67Green或PKH26Red亲脂性染料加入到500μl稀释剂中,并在黑暗中RT执行所有后续步骤。
    3. 将含有染料和稀释剂C的管与含有外来体的管或PBS中的稀释剂C和SW41Ti超速离心管中的稀释液C组合。通过手动移液快速混合。在室温下在黑暗中孵育5分钟,并在5分钟孵育期间涡旋两次。
    4. 孵育5分钟后,将含有染料,外来体或对照的超离心管和Diluent C以1:1的比例添加到无离子的FBS中孵育1分钟,以占据未结合的染料。
    5. 将溶液以100,000×g超速离心30分钟。丢弃上清液。
    6. 将含有染料标记的外来体或对照物的沉淀重悬在PBS中,转移到另一个SW41Ti超速离心管中,并以100,000×g超速离心洗涤三次,每次洗涤30分钟。每次洗涤后,将PBS再悬浮的颗粒转移到新的SW41Ti超速离心管中,以尽量减少未结合染料的转移。应使用一定体积的PBS,以完全填充超速离心管,以避免管子塌陷。
    7. 最后洗涤后,取出上清液,并将沉淀物重新悬浮于PBS中至原体积的外来体。使用Bradford Protein Assay Kit定量染色标记的外来体或对照样品,等分,并在黑暗中存放于-80°C直至使用。

  5. 用于荧光外来体内化的细胞培养物
    1. 在组织培养皿中培养人单核细胞和淋巴细胞细胞系,在补充有10%外来物质消耗的FBS,100U/ml青霉素,100μg/ml链霉素,1mM丙酮酸钠和0.3mg/ml L-谷氨酰胺的RPMI的5% CO 培养箱中37℃
    2. 在组织培养皿中培养TZM-bl阴道上皮细胞系,在补充有10%外来物质消耗的FBS,100U/ml青霉素,100μg/ml链霉素,1mM丙酮酸钠和0.3mg/ml L-谷氨酰胺的DMEM的组织培养皿中%CO 2培养箱在37℃。
    3. 在补充有0.1ng/ml预质量的人重组表皮生长因子1-53,0.05mg/ml BPE,100U/ml青霉素和100μg/ml链霉素的角质细胞血清游离培养基中的组织培养皿中生长VK2和V428阴道上皮细胞系。
    4. 生长12个组织培养皿中的所有细胞类型。在1ml体积的每种细胞类型的正确细胞培养基中,每孔1×10 5个细胞的细胞(见注1)。
    5. 在5%CO 2培养箱中在37℃下将细胞暴露于25-100μg/ml的红色或绿色荧光外来体或对照物中0,3,6,9,12或24小时的孵育期。 SE与细胞的孵育应在细胞培养基中完成。与细胞的SE孵育的最终体积将取决于荧光标记后外来体的浓度。
    6. 在平板上的1ml PBS中洗涤TZM-bl,VK2或V428贴壁细胞三次,并使用0.5ml 0.25%胰蛋白酶-EDTA解离试剂从板上分离细胞。用DMEM中的0.5ml 10%无外来物质FBS中和胰蛋白酶。从孔中收集细胞并转移到5ml聚苯乙烯圆底管中。在4℃下以500×g离心5分钟,在1ml PBS中洗涤细胞三次。
    7. 在5ml聚苯乙烯圆底管中收集单核细胞和淋巴细胞悬浮细胞。用步骤E5所述的胰蛋白酶处理,并用RPMI中的10%无外来物质的FBS中和。在4℃下以500×g离心5分钟,在1ml PBS中洗涤细胞三次。
    8. 使用FACS分析或共焦显微镜评估细胞摄取和外来体内化动力学
      1. 对于FACS分析:
        1. 用300μl2%多聚甲醛将细胞固定在冰上15分钟。
        2. 在每次洗涤之间在PBS中以500×g离心5分钟洗涤细胞两次。将细胞沉淀重悬于300μlPBS中
        3. 使用FACSCalibur或FACSVerse流式细胞仪(BD)分析荧光,以检测在融合和摄取期间从外来体转移到细胞的PKH67Green(分别为FL-1或FITC通道)或PKH26Red(分别为FL-2或PE通道)。
        4. 使用FlowJo分析软件(TreeStar)确定细胞频率和荧光强度(图1)

          图1.通过FACS进行荧光外来体的内化 A.暴露后3和24小时,PKH67绿色标记的PBS载体或100μg/ml精液外来体(SE)的VK2细胞摄取。 B.VV28细胞摄取PK67绿色标记的PBS载体或25或100μg/ml SE在暴露后24小时。 C.Jurkat,U937和TZM-bl细胞摄取PKH26红色或PKH67绿色标记的PBS载体或100μg/ml SE在暴露后24小时。 y轴显示细胞群的前向散射值(FSC)。

      2. 对于外显子内化的共聚焦显微镜分析:
        1. 通过向12孔板的孔中加入无菌的圆形18毫米盖玻片,涂覆胶原蛋白盖玻片。向孔中加入0.5 ml的50μg/ml胶原溶液。将板在37℃孵育1小时,然后将板移动到4℃进行过夜孵育。孵育过夜后,取出胶原溶液,用PBS轻轻洗涤孔中的盖玻片三次。最后洗涤后,吸出所有PBS,并立即进入步骤E8b.ii。
        2. 在胶原涂层的显微镜盖板上方的12孔组织培养板中生长TZM-bl,VK2或V428贴壁细胞,如步骤E4所述暴露于荧光外来体,并在平板中的PBS中洗涤三次。
        3. 用2%多聚甲醛将平板上的盖玻片上的细胞用冰块固定15分钟
        4. 从盖板上取下盖玻片,并向每个盖玻片加入一滴Vectashield防褪色试剂。
        5. 在显微镜载玻片上放置盖玻片。
        6. 使用激光扫描共聚焦显微镜评估荧光红色或绿色外来体的融合,摄取和内化动力学。代表性的图像显示在麦迪逊等人,2015年。//
  6. 表面外来体标记物的检测 在这里,我们将描述SE中人CD63的检测,但是该方案可能被修改用于检测SE中其他常见的外来体表面标记物。使用来自Invitrogen的外来体 - 人CD63分离/检测试剂盒,根据制造商的说明书检测SE中的人CD63,其后获得SE:
    1. 在0.1%BSA中重悬25μg的SE至总体积为100μl。
    2. 通过涡旋30秒重悬抗CD63包被的磁珠。将20μl珠转移到微量离心管中,加入200μl0.1%BSA并涡旋洗涤珠粒。
    3. 将含有珠粒的管放在磁铁分离器上1分钟。从磁铁中取出管子之前弃去上清液。
    4. 将SE溶液加入洗涤的珠子中,并通过移液混合。
    5. 在旋转混合器中,将SE /珠溶液在4℃下孵育过夜(18-20小时)。
    6. 将SE /珠子溶液离心3-5秒。
    7. 在300μl0.1%BSA中洗涤SE结合的珠子,并通过移液混合,然后将管置于磁体上1分钟,弃去上清液。
    8. 从磁铁上取下管子。将洗涤SE结合的珠子在400μl的0.1%BSA中,并通过移液混合。
    9. 将管置于磁体上1分钟,然后从磁铁中取出上清液。
    10. 将SE结合的珠子重悬于300μl的0.1%BSA中
    11. 将100μlSE结合的珠转移到新的微量离心管中。加入5μl抗人CD63-FITC(BioLegend),并通过移液混合。
    12. 将SE界限珠和抗体在室温下在黑暗中振荡板孵育60分钟
    13. 将抗体染色的SE结合珠洗涤在300μl0.1%BSA中并通过移液混合。将管放在磁铁上1分钟,弃去上清液,然后从磁铁上取出管子
    14. 重复洗涤步骤两次,然后将SE结合的珠子重新悬浮在300μl0.1%BSA中。在FACSVerse仪器和FlowJo(Tree Star)软件(图2)分析之前,将重悬的SE结合珠转移到5ml聚苯乙烯圆底管中。


      图2. SE上的CD63表达将25μgSE或PBS载体与α-CD63包被的磁珠孵育过夜以促进结合。在用α-CD63-FITC和FACS分析染色珠粒结合的SE之前,除去未结合的SE。 O/N =一夜。 y轴显示珠的前向散射值(FSC)。

      或者,也可以使用非磁珠来检测SE中的常见的外来体标志物。在这里,我们提供使用乳胶珠检测SE中的CD63的说明。
      1. 按照制造商的说明书,将2.5ml重悬浮的,不含表面活性剂的4μm直径的醛/硫酸盐胶乳珠(Invitrogen,Molecular Probes,以下称为胶乳珠)在10ml 0.025M,pH 6.0 2 - (N-吗啉代)乙烯磺酸(MES)缓冲液,在4℃下以3,000xg离心20分钟。
      2. 第二次洗涤后,将乳胶珠重新悬浮于5ml MES缓冲液中
      3. 使用100μl抗体MHC-II MAb或在MES中以1mg/ml的浓度制备的同种型对照抗体在室温下孵育100μl乳胶珠过夜,同时轻轻搅动。
      4. 通过在4℃下以3,000×g离心20分钟,将具有共轭抗体的沉淀胶乳珠子。
      5. 去除上清液(未结合的抗体)
      6. 在1℃PBS(0.1M,pH7.2)中,在4℃下,用共轭抗体洗涤乳胶珠三次,每次3次,持续20分钟。
      7. 将乳胶珠悬浮在100μl储存缓冲液中(见配方)。
      8. 将ExoQuick纯化的外来体(100μg)与* 2×10 5抗体MHC-II或同种型对照包被的胶乳珠在100μlPBS(0.1M,pH 7.2)的最终体积中首先与15 min,然后在4℃温和搅拌下过夜。
        注意:珠粒浓度/ml不同批号。
      9. 通过用含有0.2%甘氨酸的PBS(0.1M,pH7.2)30分钟温育反应停止,使乳胶珠上的任何空位饱和。
      10. 然后将外来体和抗体结合的乳胶珠制备物在FACS洗涤缓冲液中洗涤三次(参见食谱)
      11. 然后将外来体包被的珠粒与适当浓度的与PE(BioLegend)或同种型对照抗体缀合的抗CD63在无光照下在室温下孵育1小时,然后在FACS缓冲液中三次洗涤。
      12. 通过使用FACSAria流式细胞仪(BD)和FlowJo分析软件(TreeStar)分析得到的免疫荧光(图2)。

  7. SE乙酰胆碱酯酶活性
    1. 以1:1体积比溶解2%Triton-X-100中的50μgSE。
    2. 加入5μlSE/Triton-X反应至96孔平底透明板,重复三次。
    3. 以1:1比例合并1.25mM乙酰硫胆碱氯化物(Sigma-Aldrich)和0.1mM 5,5'-二硫代双硝基苯甲酸(Sigma-Aldrich),达到每孔100μl的终体积。向每个含有外来体的孔中加入100μl混合溶液。在将此溶液加入孔中之前,请确保酶标仪达到37°C,反应将在加入后立即开始。
    4. 在37℃下以5分钟的间隔读取酶标仪上450nm处的吸光度,总共30分钟(图3)。


      图3. SE的50%SE或PBS载体的乙酰胆碱酯酶活性在Triton-X-100中裂解。 AChE活性以5分钟间隔测量,共30分钟。误差条表示标准差。

  8. SE的动态光散射(DLS)
    1. 在200μlPBS中稀释0.1mg/ml的SE,并通过DynaPro NanoStar DLS(Wyatt Technologies)在塑料比色杯中使用总体积为150μl的分析尺寸。
    2. 使用Dynamics软件完成数据分析。
    3. 使用每个外来体样本平均10次测量来确定半径,直径和%强度(图4)。


      图4. SE尺寸和浓度估算。 A.动态光散射表示SE的近似直径。使用0.1mg/ml的SE来测量SE的直径。显示群体中外来体直径的范围。 B.来自NanoSight NTA视频剪辑的代表性图像,显示SE颗粒。 C.从NanoSight估计浓度计算出的每ml精液中SE颗粒的近似值。 D.通过NanoSight近似SE尺寸。 N =指外源体纯化组合供体的数量
  9. SE NanoSight纳米粒子跟踪分析(NTA)
    1. 制备含有均匀尺寸(100或200nm)聚苯乙烯颗粒的对照悬浮液。
    2. 使用悬浮液对齐激光和显微镜的焦点。
    3. 将每个SE标本在PBS中连续稀释至最终体积为0.5ml。
    4. 使用1ml一次性注射器将0.5ml稀释的SE注入NanoSight LM10 NTA。
    5. 分析所制备的连续稀释液的各个成员,直到检测到的原始浓度在仪器的推荐范围内
    6. 使用识别的稀释度,记录每个样本的三个30秒的视频。
    7. 使用相同的结算重复分析3次,以确保可重复和准确的测量。
    8. 使用NTA软件完成收购后分析,以确定SE的规模和集中度。
    9. 查看结果选项卡i)跟踪的粒子总数,ii)平均粒子数,以及iii)粒子浓度。
    10. 计算每毫升精液的外来体总浓度,考虑用于NanoSight分析的稀释因子(图4)。通过分析每个样品的3个测量的标准偏差来获取误差条
  10. SE分为膜和管腔内容物
    1. 用5-10x体积的0.1M碳酸钠pH = 11.5处理100μgSE。混合混合。
    2. 在4℃下孵育30分钟至1小时。在这一点上,来自裂解的外来体的总蛋白质可以通过NanoDrop分光光度计在280nm测量。完成以下步骤将裂解的SE分离成膜和腔分数。
    3. 将SE /碳酸钠混合物转移到SW60 Ti超速离心管中。用PBS填充体积填充管。
    4. 超声离心机在150,000xg下于4℃下1.5-2小时。沉淀物含有膜级分,而上清液含有腔内部分。
    5. 取出管腔"上清液"级分。这可以通过Bradford测定进行蛋白质定量后的MW滤光片截留浓度,并在-80℃下进行下游分析或储存直至使用。
    6. 在裂解前将PBS中的膜级分重悬于SE的原始体积。通过Bradford测定法定量蛋白质,等分,并存储在-80°C或用于下游分析(图5)。


      图5. SE分离为二 不同的级分。 SE可以分成膜和腔内成分。

  11. SE蛋白质足迹
    1. 在20μlPBS中以3μlNuPAGE溶解样品缓冲液(见Recipes)/7μlSE的比例溶解5μgSE。
    2. 在90℃的样品缓冲液中加热SE 10分钟。
    3. 在10孔1.5毫米NuPAGE 4-12%Bis-Tris凝胶上加载40μl最大体积。
    4. 在NuPAGE 20x MOPS SDS运行缓冲液稀释至1倍的条件下,以200 V运行凝胶约50分钟
    5. 银根据制造商的协议(Pierce银色染色试剂盒Thermo Scientific)对凝胶进行染色。

  12. 检查SE的完整性和内容
    1. 使用至少12μgSE的起始浓度进行RNA提取。
    2. 使用Qiagen RNeasy试剂盒根据制造商的说明书提取SE RNA,并使用Qiagen RNase-free DNA酶装置完成可选的DNA酶处理。
    3. 用NanoDrop分光光度计测定RNA浓度。为了评估SE的RNA完整性,可以通过使用RNA 6000 Pico芯片的Agilent BioAnalysis运行来分析RNA,如果RNA浓度< 50 ng /μl。如果RNA浓度> 50但< 500 ng /μl,RNA可以使用RNA 6000纳米芯片进行评估(图6)

      图6. SE的RNA完整性。从SE提取RNA,并通过Agilent Bioanalyzer分析。

    4. 使用高容量cDNA逆转录试剂盒(ABI)使用等效浓度的RNA进行cDNA合成。
    5. 使用人类基因特异性引物,通过Quantifast Sybr绿色技术(QIAGEN)和7500快速实时机器扩增CD9,CD63和GAPDH或其他感兴趣的基因。
    6. 通过溴化乙锭染色在2%琼脂糖凝胶上显示PCR扩增子。代表性的图像显示在Madison等人,2014年。

  13. SE抑制HIV-1感染性
    1. 在补充有10%外来物质消耗的FBS,100U/ml青霉素,100μg/ml链霉素,1mM丙酮酸钠和0.3mg/ml L-谷氨酰胺的DMEM的DMEM培养皿中,在96孔组织培养皿中培养TZM-bl细胞, CO 培养箱中37℃
    2. 将含有8 RT单位HIV-1病毒的100μg/ml SE或PBS载体在37℃下在补充有10%外来物质消耗的FBS,100U/ml青霉素,100μg/ml链霉素,1mM钠的DMEM中预孵育100μg/ml SE或PBS载体丙酮酸和0.3mg/ml L-谷氨酰胺。用PBS载体预培养补充的DMEM作为未感染对照。通过涡旋孵育前混合。
    3. 从TZM-bl细胞中去除DMEM培养基。加入100μl每孔一次三份SE/HIV-1,PBS载体/HIV-1或PBS载体/DMEM至TZM-bl细胞。
    4. 在5%CO 2培养箱中37℃孵育24小时。
    5. 通过MTT测定和通过Steady-Glo荧光素酶测定法检测HIV-1感染性的细胞活力(参见附注5;图7)
      1. 对于MTT测定:
        1. 在每个孔中加入20μl5mg/ml的MTT试剂,重复三次。在37℃的5%CO 2培养箱中孵育3.5小时。
        2. 从孔中取出介质和MTT溶液。向每个孔中加入150μlMTT溶剂(参见食谱),并在振荡板上在黑暗中孵育15分钟。
        3. 在酶标仪上读取590 nm的吸光度。
      2. 对于稳定的荧光素酶测定:
        1. 从TZM-bl细胞中去除DMEM培养基。在每个孔中加入100μlSteady-Glo荧光素酶试剂,重复三次,包括未感染的对照。允许细胞裂解5分钟。
        2. 将90μl裂解细胞/Steady-Glo混合物转移到固体白色96孔板中,避免形成气泡。
        3. 在微孔板光度计中读取荧光素酶活性。


          图7. HIV-1的SE抑制将外周血(100μg/ml)或载体PBS与8个RT单位HIV-1 NL4.3病毒在37℃预孵育1小时,然后感染TZM-bl细胞24h。 A.荧光素酶单位测量的感染率B.生存力由MTT确定。在用8个RT单位HIV-1 NL4.3病毒感染另外24小时之前,用外来体(100μg/ml)或载体PBS预处理TZM-bl细胞24小时。 C.荧光素酶单位读取的感染率D. MTT测定的活力。车辆设置为100%的传染性和可行性参考。错误栏是标准差。

数据分析

  1. 每个实验应通过实验重复至少三次,每次实验重复三次以验证重现性。
  2. 平均复制独立实验评价统计学意义。在适当的情况下,将结果与车辆控制进行比较
  3. 使用GraphPad软件绘制数据,如Graphpad Prism。对于未以图形方式表示的分析,如Bioanalyzer,蛋白质足迹和FACS分析,可以显示代表性的图像。

笔记

  1. 细胞培养
    当生长细胞时,为了最小化蒸发,仅在板的内孔中铺板,并用PBS填充最外层的孔。我们建议在使用前将细胞电镀,以使细胞正常化。因为SE会以不同的浓度和细胞类型依赖的方式改变细胞存活力,所以在涉及使用SE的细胞治疗的所有实验中评估细胞的细胞毒性是重要的(Madison等人,2014; Madison 等,,2015)。当评估HIV-1感染性时,这也很重要,因为高浓度的病毒或SE可能对细胞具有细胞毒性并影响结果分析。
  2. 外来体存储
    我们发现外来体反复冻融降低了功能活性。我们建议隔离后,将外来体等分成各个微量离心管(<100μg),然后冷冻保存以保留功能活性。
  3. 丢弃上清液
    当造粒或洗涤外来体后丢弃上清液时,我们建议通过移液管抽吸而不是倒置来去除,以确保完全去除。然而,根据使用的浓度,外来物质颗粒可能不总是容易观察到,并且应谨慎使用以不会使颗粒物移动。
  4. 无菌
    经常可行的是,所有实验步骤都应在层流罩下完成,以确保无菌环境。污染可能影响下游分析。
  5. HIV-1感染性
    因为TZM-bl细胞含有背景荧光,因此我们建议以96孔格式每孔铺装10,000个细胞,以使背景荧光素酶表达最小化。根据所使用的细胞系,使用的HIV-1分离物可以根据该细胞系上表达的受体/辅助受体而变化。如果评估不是报告细胞系的细胞感染,可以使用HIV-1基因特异性引物通过qRT-PCR评估感染。数据可以通过测量细胞内或细胞外HIV-1 RT活性来确认(Madison等人,2014; Madison等人,2015)。所有HIV-1实验必须按照生物安全培训和实验室环境要求进行。
  6. SE介导的HIV-1抑制
    SE还可以在感染预处理模型中抑制HIV-1感染。 在该模型中,生长细胞后,SE在病毒感染前24小时加入到细胞中,并保持对感染的抑制。 在其他细胞系如Jurkat,SUPT1,U937,PM1,THP-1,CEM和PBLs的预温育和预处理模型中,SE的HIV-1抑制特征是坚持的(Madison等人 >,2014; Madison 等人,2015)。
  7. 流式细胞仪规格
    本协议中使用的流式细胞仪系统的激光和滤波器规格如下。
    FACSCalibur流式细胞仪(BD)
    激光器:空气冷却,氩离子,488nm,15mW
    排放检测:
                 FL1 530/30
                 FL2 585/42
                 FL3 670 LP
                 FL4 661/16
                 SSC 488/10
                 FSC 488/10
    FACSVerse流式细胞仪(BD)
    Lasers:  Blue laser, 488 nm, 20 mW, beam spot size 9 x 63 μm
                 Red laser, 640 nm, 40 mW, beam spot size 9 x 63 μm
                 Violet laser, 405 nm, 40 mW, beam spot size 9 x 63 μm
    Emission detection:
                 FITC 527/32
                 PE 586/42
                 PerCp 700/54
                 APC 660/10
                 SSC 488/15
                 FSC 488/10
    FACSAria流式细胞仪(BD)
    激光器:  相干蓝宝石,固态,488nm,20mW
                JDS Uniphase HeNe, air-cooled, 633 nm, 18mW
                Point Source Violet, solid state, 405 nm, 15 mW
    Emission detection:
                 FITC 530/30
                 PE 576/26
                 PerCp 695/40
                 APC 660/20
                 SSC 488/10
                 FSC 488/10

食谱

  1. 外来体消耗的FBS
    使用SW32Ti转子的SW32Ti超速离心管中,在4℃下使用100,000 x g的超离心机FBS 2小时 收集上清液,并在4℃下储存长达一周或-80℃保存更长时间
  2. 用于蛋白质足迹的裂解样品缓冲液
    将250μlNuPAGE 4x LDS样品缓冲液与100μlNuPAGE 10x还原剂混合 向SE样品添加混合缓冲液,每7μlSE 3μl缓冲液
  3. 存储缓冲区
    PBS(0.1M,pH7.2)和0.1%甘氨酸
  4. MES缓冲区
    溶解浓度为0.025 M的蒸馏水中的MES。用pH计测定pH值,并用1N NaOH调节pH至6.0。
  5. FACS洗涤缓冲液
    PBS中的1%外来体缺失的FBS
  6. MTT试剂
    按照制造商的说明书
    重新悬浮MTT试剂至PBS中,最终浓度为5 mg/ml
  7. MTT溶剂
    0.1%NP-40和4mM HCl的异丙醇溶液

致谢

这项工作得到了国家药物滥用研究所(NIDA)授权1R01DA042348-01(CMO),美国国立卫生研究院(NIH)5T32AI007533-18(至JLW)和NIH T32传染病(MNM)博士后培训资助),共享仪表授权1S10RR025439-01到爱荷华大学中央显微镜核心设施,霍顿全面癌症中心支持授权P30CA086862。作者感谢爱荷华大学的Aloysius Klingelhutz提供V428细胞,给田纳西州中部生殖专业实验室的Bartholomey Konan和Melanie Freeman以及Amy E.T.爱荷华大学医院和诊所(UIHC)体外受精和生殖检测实验室的火花,用于提供预先存在的,未确定的人体供体精液样品。我们承认爱荷华大学核心设施的支持,包括中央显微镜,X射线晶体学和DNA核心。我们感谢爱荷华大学晶体学核心设施的Sankar Baruah和Lokesh Gakhar帮助动态光散射。作者宣称他们没有竞争的利益。 MNM,JLW和CMO写了这篇文章。 MNM和JLW对这份手稿作出了同样的贡献,因此是第一作者。所有作者审查了稿件并批准了最终版本。本文中描述的协议是从我们之前发表的作品(Madison等人,2014和2015; Welch等人,2017年)中改编的。

参考文献

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引用:Madison, M. N., Welch, J. L. and Okeoma, C. M. (2017). Isolation of Exosomes from Semen for in vitro Uptake and HIV-1 Infection Assays. Bio-protocol 7(7): e2216. DOI: 10.21769/BioProtoc.2216.
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